The opioid crisis highlights the urgent need for novel non-addictive pain medications, as well as improved treatments for opioid addiction and overdose. There is a need for pharmacological agents directed at novel targets to test new therapeutic hypotheses, new clinic-ready drugs directed at those targets, and new testing systems with the potential to be more predictive of human clinical response than traditionally used models. This initiative proposes to develop and employ novel chemistry, screening, and testing methodologies to discover new pharmacological tools and investigational drugs for pain, addiction, and overdose. Remarkable advances in our understanding of the molecular and neural circuit physiology of pain, addiction, and overdose have suggested many putative approaches for therapeutic intervention. Very few of these are considered validated; most lack sufficient data to merit the considerable resources required to develop a new drug. Lack of validation is due largely to a combination of absence of drug-like pharmacological probes for novel targets which allow testing of new therapeutic hypotheses, lack of preclinical models sufficient to reliably predict human responses, and lack of market attractiveness (for treatments for addiction and overdose). This project intends to de-risk novel targets for pain, addiction, and overdose by creating preclinical pharmacological probes for novel targets. NCGC will utilize its highly diverse small molecule libraries, including a comprehensive collection of approved drugs, a collection of over 2,000 compounds that represent diverse mechanisms, a large collection of over 8,000 pharmacologically annotated investigational drugs including tool compounds that target epigenetic modulators, a comprehensive screening collection of >200,000 drug-like small molecules, and several collections of purified natural products including those from NCI and FIC. NCATS has multiyear experience performing quantitative high-throughput screening (qHTS) that enables generation of dose response curves for every compound, thus providing unique and rich datasets. Using cheminformatics tools and counterscreen/confirmatory assays, NCGC medicinal chemists will collaborate with partnering investigators to optimize promising compounds to improve potency, selectivity, and pharmacokinetic properties. These probe compounds will be further optimized through testing in advanced efficacy assays, with a view to moving these leads closer to the clinic as future therapies for pain, addiction, and overdose.